Until quite recently, the consideration of oligonucleotides in any function other than strictly informational was unheard of. Despite the fact that certain oligonucleotides were known to have interesting structural possibilities (e.g., t-RNAs) and other oligonucleotides were bound specifically by polypeptides in nature, very little attention had been focussed on the non-informational capacities of oligonucleotides. For this reason, among others, little consideration had been given to using oligonucleotides as pharmaceutical compounds.
There are currently at least three areas of exploration that have led to serious studies regarding the use of oligonucleotides as pharmaceuticals. In the most advanced of the fields, antisense oligonucleotides are utilized to bind to certain coding regions in an organism to prevent the expression of proteins or to block various cell functions. The discovery of RNA species with catalytic functions--ribozymes--has led to the consideration of RNA species that serve to perform intracellular reactions that will achieve desired effects. And lastly, the discovery of the SELEX process (Systematic Evolution of Ligands by Exponential Enrichment) has shown the research community that oligonucleotides can be identified that will bind to almost any biologically interesting target.
The use of antisense oligonucleotides as a method for controlling gene expression and the potential for using oligonucleotides as pharmaceutical materials has prompted investigations into the introduction of a number of chemical modifications into oligonucleotides to increase their therapeutic activity. Such modifications are designed to increase cell penetration of the oligonucleotides, to stabilize them from nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide analogs in the body, to enhance their binding to targeted RNA, to provide a mode of disruption (terminating event) once sequence-specifically bound to targeted RNA, and to improve their pharmacokinetic properties. For example, PCT Patent Application Publication WO 91/14696, entitled: Oligonucleotide-Transport Agent Disulfide Conjugates, describes a method for chemically modifying antisense oligonucleotides to enhance entry into a cell.
A variety of methods have been used to render oligonucleotides resistant to degradation by exonucleases. PCT Patent Application Publication WO 90/15065, entitled: Exonuclease-Resistant Oligonucleotides and Methods for Preparing the Same, describes a method for making exonuclease-resistant oligonucleotides by incorporating two or more phosphoramidite and phosphoromonothionate and/or phosphorodithionate linkages at the 5' and/or 3' ends of the oligonucleotide. PCT Patent Application Publication WO 91/06629, entitled: Oligonucleotide Analogs with Novel Linkages, describes oligonucleotide compounds with one or more phosphodiester linkages between adjacent nucleotides replaced by a formacetal/ketal type linkage which are capable of binding RNA or DNA.
A common strategy for stabilization of RNA against endonucleolytic cleavage is to modify the 2'-position of ribonucleotides. One approach to stabilization against base-specific endonucleolytic cleavage rests on the interference with base recognition by enzymes. Several strategies for this modification are known, including modification with 2'-amino and 2'-fluoro (Hobbs et al. (1973) Biochemistry 12:5138; Guschlbauer et al. (1977) Nucleic Acids Res. 4:1933), and 2'-OCH.sub.3 (Shibahara et al. (1987) 15:4403; Sproat et al. (1989) Nucleic Acids Res. 17:3373). PCT Patent Application Publication WO 91/06556, entitled: 2' Modified Oligonucleotides, describes nuclease-resistant oligomers with substituents at the 2' position. PCT Patent Application Publication WO 91/10671, entitled: Compositions and Methods for Detecting and Modulating RNA Activity and Gene Expression, describes antisense oligonucleotides chemically modified at the 2' position and containing a reactive portion capable of catalyzing, alkylating, or otherwise effecting the cleavage of RNA, a targeting portion, and a tether portion for connecting the targeting and reactive portions.
The 5-position of pyrimidines may also be chemically modified. The introduction of modifications at the C-5 position of pyrimidines may be envisioned to interfere with the recognition by pyrimidine specific endonucleases. However, this concept is not as clear cut as the modification of the 2'-position of ribonucleotides. The current understanding of the substrate recognition of pyrimidine specific nucleases, based on X-ray studies, postulates that O.sub.4 and N.sub.3 of the pyrimidines serve as hydrogen bond contact points (FIG. 1) (Takenaka et al. (1984) Nucleic Acids Symp. Ser. 15:113). Even the modified purine 8-oxo-guanosine-2'-monophosphate, which can provide these two contacts, serves as a tight binding inhibitor (Borkahoti and Palmer (1983) J. Mol. Biol. 169:743).
Recent research has shown that RNA secondary and tertiary structures have important biological functions (Tinoco et al. (1987) Cold Spring Harb. Symp. Quant. Biol. 52:135; Larson et al. (1987) Mo. Cell. Biochem. 74:5; Tuerk et al. (1988) Proc. Natl. Acad. Sci. USA 85:1364; Resnekov et al. (1989) J. Biol. Chem. 264.:9953). PCT Patent Application Publication WO 91/14436, entitled: Reagents and Methods for Modulating Gene Expression Through RNA Mimicry, describes oligonucleotide or oligonucleotide analogs which mimic a portion of RNA able to interact with one or more proteins. The oligonucleotides contain modified internucleoside linkages rendering them nuclease-resistant, have enhanced ability to penetrate cells, and are capable of binding target oligonucleotide sequences.
Palladium-catalyzed reactions involving organotin compounds have been explored for many years. These reactions constitute some of the best ways to form new carbon-carbon bonds. The reactions are generally characterized as substitution or addition reactions between an organotin species and an organic electrophile. ##STR1## When the electrophile is an acyl halide, or similar species, carbonylation substitution occurs as follows: ##STR2##
Carbonylative substitution may also be accomplished using an alkyl halide, carbon monoxide and an organotin species in the presence of a palladium catalyst. ##STR3## Early reviews of palladium-catalyzed substitution and addition reactions by Stille and Mitchell provide a great deal of information regarding the scope of the reaction and variations that are acceptable and desirable in certain situations. See, Stille (1986) Angew. Chem. 98:504; Angew. Chem. Inst. Ed. Engl. (1986) 25:508; Mitchell (1986) J. Organomet. Chem. 304:1. A more recent review of palladium-catalyzed reactions of organotin compounds is also available. See, Mitchell (1992) Angew. Chem. Int. Ed. Engl. 9:803-815.
Although there has been a great deal of effort in studying the palladium-catalyzed reactions, extension of the system to new reactions is not always straightforward. As stated recently by Professor Mitchell when reviewing the recent literature, "particular attention will be paid to catalyst variations, since the choice of the right catalyst for a particular task often appears from the literature to involve a fair degree of alchemy", Mitchell supra.
The use of palladium to catalyze carbon-carbon bond formation at the 5 position of pyrimidine nucleosides is not unknown. The first use of this technique was demonstrated by Bergstrom (Bergstrom et al. (1976) J. Am. Chem. Soc. 98:1587, (1978) J. Org. Chem. 43:2870, (1981) J. Org. Chem. 46:1432 and 2870, (1982) J. Org. Chem. 47:2174) and Daves (Arai and Daves (1978) J. Am. Chem. Soc., 100:287; Lee and Daves (1983) J. Org. Chem. 48:2870). Bergstrom and Daves used 5-mercurial-deoxyuridine compounds, the same as those used by Dreyer and Dervan ((1985) Proc. Natl. Acad. Sci. USA 82:968) to tether functional groups to oligonucleotides.
One method for simple carbon-carbon coupling reactions to the 5-position of uridines is described in the work of Crisp (1989) Syn. Commun. 19:2117. Crisp forms deoxyuridines functionalized at the 5 position by reacting protected 5-iodo-2'-deoxyuridine with alkenylstannanes in acetonitrile in the presence of a Pd (II) catalyst. Crisp's protocol differs from that of the present invention in three important ways. First, it requires acetonitrile as solvent and uses PdCl.sub.2 (PPh.sub.3).sub.2 as catalyst. This catalyst does not work well when THF is used as the solvent. Second, the Crisp catalyst has less general applicability, being incapable of facilitating the coupling of aromatic groups. Thirdly, the prior art methods required protection/deprotection schemes. Further, independent attempts to repeat literature procedures involving Pd(II) species have shown that the results were not reproducible. In a later paper [(1990) Tetrahedron Lett. 31:1347] Crisp used the 5-triflate uridines to react with the organostannanes, to synthesize 5-aryl and 5-vinyl uridine analogs. The 5-triflate uridine starting materials are very difficult to prepare.
SELEX (Systematic Evolution of Ligands for Exponential Enrichment) is a method for identifying and producing nucleic acid ligands, termed "nucleic acid antibodies", e.g., nucleic acids that selectively bind to target molecules (Tuerk and Gold (1990) Science 249:505). The method involves selection from a mixture of candidates and step-wise iterations of structural improvement, using the same general selection theme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, the method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound to target molecules, dissociating the nucleic acid-target pairs, amplifying the nucleic acids dissociated from the nucleic acid-target pairs to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired.
The methods of the present invention may be combined with SELEX to produce nucleic acid antibodies containing modified nucleotides. The presence of modified nucleotides may result in nucleic acid antibodies with an altered structure exhibiting increased capacity to bind target molecules. The steric and electronic influence of 5-position modified nucleotides may also act to prevent nuclease degradation.